PubMed Health⌕ Search

Biomedical subjects

S Jackowski

Publications and source records attributed to S Jackowski.

At least 37 records · Page 2Linked to original sources
PubMed1997

The role of CDP-diacylglycerol synthetase and phosphatidylinositol synthase activity levels in the regulation of cellular phosphatidylinositol content.

The regulation of phosphatidylinositol synthesis was examined by cloning and expressing in COS-7 cells the human cDNAs encoding the two enzymes in the biosynthetic pathway. Human CDP-diacylglycerol synthetase (cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequence-tagged (EST) data base, and full-length cDNAs were obtained by library screening. The cds1 cDNA did not possess a recognizable mitochondrial import signal, and the activity of the expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme. There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues. Consistent with the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the EST data base. In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined. Expression of the pis1 gene led to the overproduction of both phosphatidylinositol synthase and phosphatidylinositol:inositol exchange reactions, indicating that the Pis1 polypeptide catalyzed both of these activities. Phosphatase treatment of cell extracts abolished the CMP-independent phosphatidylinositol:inositol exchange reaction, and exchange activity was completely restored by the addition of CMP. Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phosphatidylinositol biosynthesis. Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diacylglycerol or phosphatidylinositol. These data illustrate that the levels of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for the activity of either of these enzymes in the regulation of PtdIns biosynthesis.

Amino Acid Sequence↗
PubMed1997

Phosphatidylcholine signaling in response to CSF-1.

The formation of cell membrane following CSF-1 stimulation of a macrophage cell line is coordinated with cell cycle progression. The majority of membrane phospholipid accumulates during the S phase and results from cell-cycle dependent oscillations in the rates of phosphatidylcholine biosynthesis and degradation. Both synthesis and degradation are enhanced during the G1 phase, resulting in a high rate of phosphatidylcholine turnover. Degradation of phosphatidylcholine after CSF-1 stimulation is mediated by a phospholipase C, and the release of diacylglycerol during G1 phase is biphasic. The degradation essentially stops during the S phase, thus allowing biosynthesis to supply the necessary membrane for cell division and doubling. The degradation of phosphatidylcholine during G1 signals the downstream activation of c-fos and junB transcription and can be mimicked by incubation of the macrophage cells with exogenous bacterial phospholipase C. In contrast, the expression of c-myc transcripts normally associated with CSF-1 stimulation is severely compromised in phospholipase C-treated cells, indicating that the diacylglycerol signals a pathway distinct from the pathway that governs c-myc activation. Constitutive expression of c-myc complements phospholipase C activity and permits the growth of cells in the presence of exogenous bacterial enzyme and the absence of CSF-1. Protein kinase C is not required to mediate the diacylglycerol signal that supports cell growth. GTP exchange on Ras is not enhanced, and MAP kinase activity is not stimulated in response to phosphatidylcholine degradation by exogenous phospholipase C. The 85 kDa cytoplasmic phospholipase A2 is activated, however, as well as a novel protein we have called p96. Rapid serine phosphorylation of p96 follows stimulation of cells with either CSF-1 or exogenous phospholipase C. Analysis of the murine cDNA encoding p96 reveals an amino-terminal domain with significant similarity to the amino-terminal domain of the Drosophila-disabled gene product and a carboxy-terminal domain containing proline-rich sequences characteristic of SH3 binding regions. The sequence of p96 suggests an interactive role for this unique protein in the CSF-1 signal transduction cascade.

Animals↗
PubMed1996

Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs. Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42 degrees C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane. Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids.

Alleles↗
PubMed1995

The association of lipid activators with the amphipathic helical domain of CTP:phosphocholine cytidylyltransferase accelerates catalysis by increasing the affinity of the enzyme for CTP.

The biochemical mechanism for the regulation of enzyme activity by lipid modulators and the role of the amphipathic alpha-helical domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated by analyzing the kinetic properties of the wild-type protein and two truncation mutants isolated from a baculovirus expression system. The CT[delta 312-367] mutant protein lacked the carboxyl-terminal phosphorylation domain and retained high catalytic activity along with both positive and negative regulation by lipid modulators. The CT[delta 257-367] deletion removed in addition the region containing three consecutive amphipathic alpha-helical repeats. The CT[delta 257-367] mutant protein exhibited a significantly lower specific activity compared to CT or CT[delta 312-367] when expressed in either insect or mammalian cells; however, CT[delta 257-367] activity was refractory to either stimulation or inhibition by lipid regulators. Lipid activators accelerated CT activity by decreasing the Km for CTP from 24.7 mM in their absence to 0.7 mM in their presence. The Km for phosphocholine was not affected by lipid activators. The activity of CT[delta 257-367] was comparable to the activity of wild-type CT in the absence of lipid activators and the CTP Km for CT[delta 257-367] was 13.9 mM. The enzymatic properties of the CT[delta 231-367] mutant were comparable to those exhibited by the CT[257-367] mutant indicating that removal of residues 231 through 257 did not have any additional influence on the lipid regulation of the enzyme. Thus, the region between residues 257 and 312 was required to confer lipid regulation on CT, and the association of activating lipids with this region of the protein stimulated catalysis by increasing the affinity of the enzyme for CTP.

Animals↗
PubMed1995

Lipid activation of CTP:phosphocholine cytidylyltransferase is regulated by the phosphorylated carboxyl-terminal domain.

The role of the phosphorylated carboxyl-terminal domain of CTP:phosphocholine cytidylyltransferase (CT) in the regulation of enzyme activity was investigated by comparing the catalytic properties of wild-type CT to two mutant proteins with altered carboxyl-terminal phosphorylation domains. CT isolated from a baculovirus expression system was extensively phosphorylated at multiple sites in the carboxyl-terminal domain. The CT[S315A] mutant lacked a major CT phosphorylation site, and the carboxyl-terminal deletion mutant, CT[delta 312-367], was not phosphorylated. The higher activities of CT[delta 312-367] and CT[S315A] relative to CT were attributed to differences in the sensitivities of the enzymes to lipid activators. The rank order of the apparent Km values for activation by either phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol was CT > CT[S315A] > CT[delta 312-367]. In addition, CT exhibited negative cooperativity in its activation by phosphatidylcholine/oleic acid (nH = 0.64) and phosphatidylcholine/diacylglycerol (nH = 0.74) vesicles, whereas CT[delta 312-367] and CT[S315A] did not. These data support the concept that the phosphorylation of the CT carboxyl-terminal domain interferes with the activation of CT by lipid regulators.

Animals↗
PubMed1995

Cloning of a novel phosphoprotein regulated by colony-stimulating factor 1 shares a domain with the Drosophila disabled gene product.

A unique protein with an apparent molecular mass of 96 kilodaltons (p96) was detected in the murine macrophage cell line, BAC1.2F5. The murine cDNA encoding p96 was cloned and sequenced, along with cDNAs representing two alternatively spliced forms of the protein. All three proteins possessed identical amino-terminal domains with significant similarity to the amino-terminal domain of the Drosophila disabled gene product and carboxyl-terminal domains containing proline-rich sequences characteristic of src homology region (domain 3) binding regions. BAC1.2F5 cells predominantly expressed the p96 protein, although mRNA and protein corresponding to the p67 splice variant were also detected. Electrophoretic gel retardation of p96 in response to stimulation of the cells with colony-stimulating factor 1 was noticeable within 5 min after growth factor addition and reached a maximum at 60 min. Metabolic labeling experiments showed that the gel retardation of p96 was associated with increased phosphorylation of the protein exclusively on serine residues. These data identify a novel protein that is phosphorylated in response to mitogenic growth factor stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Deregulated c-myc expression overrides IFN gamma-induced macrophage growth arrest.

Induction of c-myc gene expression is an essential response to growth promoting agents, including colony-stimulating factor 1 (CSF-1). Down regulation of c-myc expression occurs in response to a variety of negative growth regulators in many cell types. However, for many of these systems the causal link between c-myc down regulation and growth arrest remains to be established. Here we show for CSF-1-dependent BAC1.2F5 mouse macrophages that interferon-gamma (IFN gamma) results in a midlate G1 phase decrease of CSF-1-dependent c-myc mRNA and subsequent cell cycle arrest. Introduction of a deregulated c-myc gene into these cells, which prevents the IFN gamma-mediated decrease in c-myc expression, overrides the cell cycle arrest and restores CSF-1-dependent growth in the presence of the cytokine. This result contrasts with the macrophage growth arrest induced by cAMP elevation, which also suppresses c-myc expression, but is not overcome by a deregulated c-myc gene. These results show that inhibition of c-myc expression is an essential component in IFN gamma-mediated cell cycle arrest and demonstrates that distinct mechanisms contribute to IFN gamma- and cAMP-mediated growth arrest in macrophages.

Animals↗
PubMed1995

Lysophosphatidylcholine attenuates the cytotoxic effects of the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3- phosphocholine.

A colony-stimulating factor 1-dependent cell line was used to determine the relationship between the inhibition of phospholipid synthesis and the cytotoxic activity of the antineoplastic ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). ET-18-OCH3 inhibited choline incorporation into phosphatidylcholine as well as total phospholipid synthesis. Exposure to ET-18-OCH3 at the G1/S boundary led to the accumulation of cells in G2, whereas the addition of ET-18-OCH3 in the G1 phase of the cell cycle prevented entry into the S phase. In both cases, ET-18-OCH3 treatment triggered DNA fragmentation and morphological changes associated with apoptosis within 10 h. The addition of lysophosphatidylcholine provided an exogenous source of cellular phospholipid and prevented ET-18-OCH3-dependent accumulation of cells in G2 and apoptosis. However, lysophosphatidylcholine did not overcome the ET-18-OCH3-dependent G1 block, although the growth-arrested cells remained viable. These data indicate that restoring phosphatidylcholine synthesis by supplementation with lysophosphatidylcholine overrides the cytotoxic but not the cytostatic activity of ET-18-OCH3.

Animals↗
PubMed1995

Lysophosphatidylcholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step.

The regulation of the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step by lysophosphatidylcholine (LPC) and the nonhydrolyzable LPC analog, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was investigated in a colony-stimulating factor 1-dependent murine macrophage cell line. LPC inhibited phosphatidylcholine synthesis in vivo and led to the accumulation of choline and phosphocholine coupled to the disappearance of CDP-choline pointing to CT as the intracellular target. LPC neither inhibited cell growth nor decreased the cellular content of CT or altered the distribution of CT between soluble and particulate subcellular fractions. The inhibition of phosphatidylcholine synthesis was specific for LPC since lysophospholipids lacking the choline headgroup were not inhibitors. ET-18-OCH3 was a more potent inhibitor of phosphatidylcholine synthesis than LPC and caused the translocation of CT from the soluble compartment to the particulate compartment. Both LPC and ET-18-OCH3 were inhibitors of CT activity in vitro and kinetic analysis showed competitive inhibition with respect to the lipid activator. These data point to LPC as a negative regulator of de novo phosphatidylcholine synthesis that acts at the CT step and establish the mechanism for the inhibition of phosphatidylcholine biosynthesis by antineoplastic phospholipids.

Animals↗
PubMed1994

Regulation of cytosolic phospholipase A2 phosphorylation and eicosanoid production by colony-stimulating factor 1.

A colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell line (BAC1.2F5) and peritoneal macrophages were used to investigate the relationship between growth factor-dependent phosphorylation/activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) and arachidonic acid metabolism. The addition of CSF-1 to quiescent BAC1.2F5 cells was followed by the rapid phosphorylation, electrophoretic gel retardation, and stable increase in the specific activity of cPLA2 that correlated with the activation of ERK kinases. cPLA2 phosphorylation depended on the presence of growth factor and persisted throughout the cell cycle. CSF-1 inhibited prostaglandin E2 production and did not enhance arachidonic acid release or increase the levels of lysophosphatidylcholine or glycerophosphocholine. Treatment of BAC1.2F5 cells with the calcium ionophore A23187 plus CSF-1 did not stimulate eicosanoid release. Instead, CSF-1 enhanced the rate of exogenous arachidonic acid incorporation into phosphatidylcholine and its subsequent transfer to phosphatidylethanolamine suggesting that higher rates of arachidonic acid acylation may contribute to the suppression of prostaglandin production. In peritoneal macrophages, ERK kinase activity was stimulated and cPLA2 was phosphorylated and activated in response to CSF-1. However, CSF-1 did not trigger eicosanoid release but did augment arachidonic acid mobilization and prostaglandin E2 production elicited by zymosan and A23187. Thus, cPLA2 phosphorylation/activation and calcium mobilization are not the only determinants for eicosanoid release, and additional components in differentiated tissue macrophages are also required.

Animals↗
PubMed1994

Kinetics and regulation of pantothenate kinase from Escherichia coli.

Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis and is regulated by feedback inhibition by CoA. Pantothenate kinase was purified to homogeneity from Escherichia coli and was shown to exist as a homodimer. Kinetic analysis indicated the presence of two ATP binding sites that exhibited positive cooperativity with a Hill coefficient of 1.46. Site-directed mutagenesis of lysine 101 to methionine (K101M) resulted in the inactivation of the enzyme, although dimer formation was not altered. The K101M mutant was unable to bind either adenosine 5'-O-(3-thiotriphosphate) or CoA, supporting the conclusion from kinetic analysis that both the substrate and inhibitor bind to the same site on the enzyme. CoA binding was not cooperative. Coexpression of the K101M mutant gene on a high copy number plasmid in the presence of a chromosomal copy of the wild-type gene resulted in the production of heterodimers between active and inactive subunits. Kinetic analysis of the chimeric heterodimers showed the absence of cooperative ATP interactions and indicated a sequential kinetic mechanism for pantothenate kinase with ATP binding first and pantothenate second. Thus, pantothenate kinase regulation involves the competitive binding of CoA to the ATP site, which blocks ATP binding at one site and prevents positive cooperative ATP binding to the second site on the dimer.

Adenosine Triphosphate↗
PubMed1994

Guanosine tetraphosphate inhibition of fatty acid and phospholipid synthesis in Escherichia coli is relieved by overexpression of glycerol-3-phosphate acyltransferase (plsB).

The accumulation of the alarmone guanosine-3',5'-bispyrophosphate (ppGpp) in response to amino acid starvation or energy source depletion mediates the coordinate inhibition of macromolecular and membrane phospholipid biosynthesis in Escherichia coli. Accumulation of ppGpp triggered by the induced expression of either the relA gene or an unregulated, truncated relA gene that encodes a constitutively active ppGpp synthetase I, inhibited both de novo fatty acid and phospholipid biosynthesis and the incorporation of exogenous fatty acids into phospholipid. ppGpp inhibition of fatty acid and phospholipid synthesis was associated with an accumulation of long-chain acyl-ACP, the end products of fatty acid biosynthesis, and substrates for the sn-glycerol-3-phosphate acyltransferase (the plsB gene product). Overexpression of the plsB gene product relieved the inhibition of fatty acid and phospholipid synthesis, prevented the accumulation of long-chain acyl-ACPs, and allowed an increase in cell size following elevation of intracellular ppGpp. However, stable RNA accumulation and cell division were still blocked by ppGpp accumulation. These data show that the sn-glycerol-3-phosphate acyltransferase mediates the ppGpp-dependent regulation of fatty acid and phospholipid biosynthesis in E. coli.

Acetates↗
PubMed1994

Coordination of membrane phospholipid synthesis with the cell cycle.

The formation of membrane phospholipid is coordinated with the cell cycle in a colony-stimulating factor 1-dependent macrophage cell line. Net phospholipid accumulation occurs as cells enter S phase and results from an interaction between cell cycle-dependent oscillations in the rates of phosphatidylcholine (PtdCho) biosynthesis and degradation. CTP:phosphocholine cytidylyltransferase (CT) is the rate-controlling step in PtdCho biosynthesis, and its activity is inhibited by cell cycle-dependent phosphorylation. CT phosphorylation is low in early G1, begins to rise in late G1, increases steadily through S and G2/M, and then declines precipitously as cells exit mitosis and reenter G1. CT activity increases in early G1 and then decreases steadily as the cells progress through late G1, S, and G2/M, reflecting the elevated phosphorylation state of the protein. Membrane phospholipid degradation is also periodic: the rate is rapid in G1, slows significantly during S phase, and accelerates again as cells reenter G1. The data support the hypothesis that CT dephosphorylation accelerates PtdCho synthesis in response to the increased membrane phospholipid degradation to maintain membrane phospholipid mass during G1 and that the periodic cessation of phospholipid degradation during S phase accounts for the transition to net membrane phospholipid accumulation. Inactivation of CT associated with hyperphosphorylation of the protein during G2/M likely plays a determining role in the cessation of net membrane accumulation prior to cell division.

Animals↗
PubMed1994

Sequence and function of the aas gene in Escherichia coli.

2-Acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthase activities are encoded by the aas gene in Escherichia coli. The aas gene was cloned, and the DNA sequence of the aas gene and the region between aas and galR established the clockwise gene order in the 61.2 min of the E. coli chromosome as aas-orf-galR-lysA-lysR-orf-araE. The aas gene consists of a single open reading frame of 2,157 base pairs predicted to encode a protein of 80.6 kDa. Strains harboring multiple copies of the aas gene overproduced both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities in vitro and had higher specific activities for the incorporation of exogenous fatty acids and lysophospholipids into the membrane in vivo. Specific expression of the aas gene yielded a protein with an apparent molecular mass of 81 kDa. Comparison of the predicted amino acid sequence of the aas gene with mammalian, yeast, and bacterial long chain acyl-coenzyme A synthetases revealed three domains of high similarity which are postulated to form the acyl-AMP binding pocket. These data verify that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are reactions carried out by the same gene product, verify the role of 2-acyl-GPE acyltransferase/acyl-ACP synthetase in the acylation of endogenous 2-acyl-GPE, and establish the product of the aas gene as the rate-limiting enzyme in the uptake and incorporation of exogenous 2-acyllysophospholipids.

Acyltransferases↗
PubMed1993

Expression of rat CTP:phosphocholine cytidylyltransferase in insect cells using a baculovirus vector.

CTP:phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in phosphatidylcholine biosynthesis. We constructed a recombinant baculovirus (bCT) containing rat CT cDNA under the control of the polyhedrin promoter. Crude cell extracts of Spodoptera frugiperda (Sf9) cells infected with bCT possessed 250-fold higher specific activities for CT compared to rat liver cytosol, and CT protein constituted 3-6% of the total cellular protein. The 42-kDa form of CT predicted from the cDNA sequence was the first immunoreactive CT protein detected at Day 2 after infection and this form continued to accumulate until Day 5. On Day 3 following infection, a 37-kDa protein immunologically related to CT began to accumulate, indicating that CT was being degraded. The active, 42-kDa form of CT was purified to homogeneity in a single step using hydroxyapatite chromatography. Antibodies raised against recombinant CT were employed to quantitatively extract and assay CT activity in mammalian cell lines. The baculovirus expression system is suitable for the preparation of large amounts of protein for investigating the structure, function, and regulation of CT.

Animals↗
PubMed1993

The gene for murine CTP:phosphocholine cytidylyltransferase (Ctpct) is located on mouse chromosome 16.

CTP:phosphocholine cytidylyltransferase is the rate-controlling enzyme in phosphatidylcholine biosynthesis and is essential for the survival of eukaryotic cells. The murine cDNA for the cytidylyltransferase was cloned and sequenced. A genomic clone was isolated and the chromosomal location of the Ctpct locus determined by Southern blot hybridization of DNAs from a panel of interspecific backcross progeny derived from matings of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice. These data place the Ctpct gene on mouse chromosome 16 between the Smst and Stf-1 genes.

Amino Acid Sequence↗